...particles. Direct measurement of lentiviral RNA is one alternative to direct p24 measurement. Like a p24 ELISA...that specific feature. Primers targeting the transgene are also beneficial as they ensure that the correct...stable cell line. While we’ve all experienced the pressure to move a project forward, transductions should...time to properly titer your virus will not only ensure that your infection is designed in the best possible...flavors, physical or functional. Physical titers measure the amount of viral particles in a sample and are...acid. Functional titers, or infectious titers, measure how many of the viral particles produced can actually...common assays for physical titer are direct p24 measurements via ELISA and qPCR for viral RNA. Measuring ...

...blot.   Total protein measurements as loading controls Total protein measurements are an accurate alternative...control measurement will take place either before or after blotting for your POI. Figure 6: Total...the most important feature of a loading control, and both total protein measurements are linear. Note the... a western blot requires a standard curve with a pure sample of your protein of interest (POI). This approach...pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive control... and the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each...experimental design and gel quantification. Figure 3: In a perfect world, protein concentration in...

...other materials have been washed away (Figure 1).      Figure 1: The steps of rAb production. Created... make sure to look up the setting for your isotype.  Antibody concentration may also be measured through...antibodies with some molecular biology and cell culture experience. Let’s go over the basics of making ... days (in mammalian cells) to collect the cell culture supernatant for further processing. It may be helpful...appropriate immobilized ligands are incubated with cell culture supernatant containing antibodies. The antibodies...your antibody in a downstream step, you’ll want to ensure that your buffer components are compatible with...have an IgG protein setting. If yours does not, be sure to set the absorbance to 280 nm (Pace et al., 1995...

... your culture should always be derived from a single colony to ensure uniformity of the culture. Sequencing...to a shared computer or server. This will ensure that future members of your lab can access and use the...depending on the number of features we need to verify. Determining which features to verify will depend on...important features that differentiate this plasmid from its predecessor. Important features may include...reaction, or about 250 amino acids worth, so make sure the feature you want to verify is within this range. Read...plasmids, such as special growth instructions (temperature, strain, media supplements), suggested sequencing...information in a binder or digital notebook and make sure you note the freezer location of every plasmid in...

...perform the IP at 4oC. When choosing the capture antibody for IP, be sure to choose one that recognizes the ...target protein/capture antibody intact. The wash step is repeated several times to ensure non-specific ...complexes, wash away unbound protein, elute and measure the target. However, the two different approaches...process of anchoring an antibody, often called the capture antibody, to agarose or magnetic beads in a way...immunoglobulin, leaving the antigen binding sites free to capture the target protein. The beads can be collected ...Agarose beads are sponge-like and vary in shape and structure. Their porous surface provides a large area for...protein interactions, and limit high throughput procedures. However, agarose beads are often a more cost-effective...

... view is excited at one time and captured in a single camera exposure. This is much faster than relying...the Harvard Center for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University...an x-ray, or a biologist imaging the smallest structures of life; the key to a great image is contrast...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished...resolution) limit of a light microscope. Due to the wave nature of light, an infinitely small point of light will...improvement in the resolution of a light microscope (see Figure 1). What microscopy technique should I use for ...live cells with fluorescently labelled, motile structures These samples are thin enough to sit within the...

...antibodies. Figure 1: Overview of using Sandwich ELISA to quantitatively measure protein concentration...format. Figure 2: Overview of using Competitive ELISA to quantitatively measure protein concentration...the plate. Figure 3: Overview of using Direct ELISA to quantitatively measure protein concentration...proteins. Figure 4: Overview of using Indirect ELISA to quantitatively measure protein concentration...competitive ELISA use antibodies coating the wells to capture antigen from a sample, whereas antigen is bound...coating the wells of a flat bottomed plate with a capture antibody that is specific to the protein you are...that also recognizes the same target protein. The capture antibody and the detection antibody recognize different...

...organism to target large or diffuse biological structures. Of primary interest here are systemic capsids...health. While it’s always helpful to start with a literature search to see what others have done, you’ll need... before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the... antibodies developed in response to the first exposure to AAV9. The exception is the X1 capsid for targeting...strains. It is therefore important to review the literature for use of a capsid in your strain of interest...to start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid...further highlighted below the table.     Figure 2: This decision tree can help guide your choice...

...elevated temperatures. In both cases, GeoCas9 outperformed SpCas9 at high temperatures. See Figure 1 for ...stearothermophilus grows at temperatures ranging from 30-74 °C, with an optimal growth temperature of 55 °C. For a... longevity. GeoCas9 is active at high temperatures Figure 1: Thermostability of SpCas9 vs GeoCas9... These Cas9 proteins don’t work well at high temperatures, so to use them in thermophiles, bacteria must... to make eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity via...CRAA (R = A or G) Spacer Length 20 nt 22 nt Temperature Range for Optimal Activity 35 - 45 °C 50 - 70...GeoCas9 RNPs were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity is expressed...

...publications really the best way to measure impact? As that Nature report also noted, papers detailing...scientific publications. Ventures like Impact Story seek to find ways to capture scholarly work that exists...scientific journals. Of course, no one measure will ever adequately capture a notion as complex as “scientific... you like to measure yours? More Reading Fenner M. "Altmetrics and Other Novel Measures for Scientific...Impact Measures" (2009) PLOS One 4(6): e6022. doi: 10.1371/journal.pone.0006022 Aragon AM. "A measure for...publish in journals with a high “impact factor,” a measure that dates back to 1975 and is based on the average...articles. Of course, a publication in Science or Nature is always nice, but in the wired world we are living...

...packaging as a single payload (Figure 4). Figure 4: ONE-GO biosensors measure the formation of Gα-GTP...uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with an enhanced...amplification of cargo, and heat treatment.  Figure 1: Left: recombined prime editing tools PE2max-...heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org...N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression...solution and when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule... inserted. B) Protein expression and labeling procedure. Adapted from Smith et al. 2024 under a CC-BY ...

...walks through the paper figure by figure. Some follow the same figure by figure organization with a different...adapt the second version (multiple presenter, figure by figure) if the primary purpose is training graduate...enjoy is discussion of current new and exciting literature. As I transitioned out of academia and away from...researchers of all varieties must stay current with literature and emerging technologies to remain competitive...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive...pieces before that first meeting takes place to ensure success. One of the most important is letting people...ready for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the...

...strategies such as the bisarsenical dye (Figure 1a) and FAP systems (Figure 1e) are inherently fluorogenic and...biological environments such as live cells and tissues (Figure 1). These flexible strategies give you the best...peptide tag could be used to label proteins in cells (Figure 1a). Other strategies that have been developed ...substrate ligand motif attached to a fluorophore (Figure 1b). Engineered ligases (e.g., lipoic acid ligase...attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–...amino acids can be incorporated into a protein structure and then used with the growing toolbox of bioorthogonal...fluorophore at the specific site of incorporation (Figure 1d). Fluorogen activating proteins (FAPs) – These...

... need to pick a fluorophore for each target and ensure the fluorophores, together, are able to give you...most efficient (i.e., happen most of the time). Figure 1 shows a graph from the BD® Spectrum Viewer, outlining...Fluorescence Resonance Energy Transfer (FRET).   Figure 1: Emission spectra and respective colors of a ... we can recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and ...laser. To understand why that is, let's look at Figure 2. The blue laser excites at a wavelength of 488...maximum of PE's excitation spectrum (dotted line, Figure 2). The yellow/green laser, however, excites at...relatively stronger — or brighter — signal.   Figure 2: Excitation and emission spectra for PE. The ...

...transcript by endogenous processing enzymes (Figure 1). To ensure this tRNA processing only occurs on the ...recommends both for use in western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-...improvements in performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor...reconstituted within the target cells (Figure 6).   Figure 6: Illustration of split fluorophore ...Zeocin®, or nuclear expression of BFP2.     Figure 1: CROPseq-multi uses two sgRNAs with internal ... that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in...just begun with PE7, it will be exciting to see future studies enabled by this tool as well as additional...

...will cover: Miniature CRISPR-Cas systems SEND plasmids Prime editing improvements Miniature CRISPR-Cas...problem. A miniature CRISPR-Cas system for mammalian use Stanley Qi’s lab has developed a miniature Cas system...paper in Nature Biotechnology Find the Cas12f-GE plasmids Programmed genome editing by a miniature CRISPR-Cas12f... guide RNAs Yong-Sam Kim’s lab also created a miniature yet active Cas system starting with the same Un1Cas12f1...nuclease Quanjiang Ji’s lab describes an effective miniature Cas system that utilizes a Cas12f from a different...ribonucleoprotein, and AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   ...paper in Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo...

...and, when they mature, fluoresce red. The ratio of the mature color (red) to the immature color (green ...determination of expression time The ratio of mature to immature fluorescence from the FP timer is dependent...their knowledge of protein structure and saturation mutagenesis. mK-GO matures from green to red, and the...they predominantly fluoresce one color but slowly mature until they predominantly fluoresce a second color...580 nm) over the course of 18 hours in vitro (see figure 1B in Terskikh et al.) and even displays predictable...linear window for the theoretical timer shown in Figure 1B, you can use the equation fit to this linear... system composed of two separate FPs (one that matures quickly and the other slowly, see Verkhusha et ...

... from a heterogeneous mixture - for example, cell lysate or media from cultured cells. ELISA can be used...cell subsets of interest from among the mixture and to measure the relative amount of your protein of ...antigen the antibody recognizes and binds to.   Figure 1: Labeled diagram of an antibody including Fc,...). Single-chain variable fragment (ScFv) Figure 2: Comparison between the IgG antibody and scFv...Chain Fragment Variables (scFvs). Nanobodies Figure 3: Comparison of the Hcab and nanobody. Nanobodies...can be used for qualitative and quantitative measurement of protein expression in cell lysates, whole ...presence of proteins from samples containing a mixture of proteins. The proteins are separated based on...

... IF instead (Figure 1B). To label all your targets in one step, you simply use a mixture of primary antibodies...puncta in each cell. When making figures for a paper or presentation, make sure the images are accessible to...as immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC) with tissues... you can perform simultaneous detection with a mixture of primary antibodies and appropriate secondary...(Im et al., 2019; McLaughlin, 2019).    Figure 1: Schematic of multiplex immunofluorescence approaches.... Sequential IF For a sequential IF experiment (Figure 1A), you’ll block, add primary antibody to bind...antibodies, wash, add a mixture of secondary antibodies, and image all the different colors in one session...

...is consistent with the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal...effect, which is green fluorescent cells (Figure 3).   Figure 3: Expression of Plasmid B (as positive control...control plasmids? Generally, control plasmids help to ensure that the observed phenomenon in your experiment...of Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown...vector and internal control A transfection control measures transfection efficiency and enables observation...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore...transfection control is an internal control vector, which measures transfection efficiency. An internal control may...